2.2.2. Oxidative Stability of Mackerel Tuna Fillets

The deterioration of the quality of fish and its products during storage is mainly due to the oxidation of lipid [11]. Lipid peroxidation is the reaction of oxygen with unsaturated lipids; hence, one of the methods that delays or prevents oxidation processes is the use of edible coatings for fish fillets [37] because the edible coatings can guarantee performance as a low oxygen barrier [24].

The peroxide value (POV) is a substantial indicator of fat rancidity, but how does fat rancidity happen? Rancidity happens through the process of lipid oxidation, which is accompanied by the production of free radicals, which in turn leads to the formation of aldehydes and ketones, all of which, of course, negatively affect the quality of fish [38]. As the storage period progressed, the peroxide values in coated and uncoated mackerel

tuna fillets increased, with the control (uncoated) samples having the highest (*p* < 0.05) peroxide value at each interval storage period (Figure 2). The peroxide value of the control (uncoated fillet samples) increased from 2.22 to 17.32 meq peroxides/kg lipid, while during this time, the peroxide value of XAN-EEP 0%, XAN-EEP 1%, and XAN-EEP 2% increased from 2.23 to 14.55, 2.18 to 9.89, and 2.25 to 8.44 meq peroxides/kg lipid, respectively, after 20 days of chilled storage. In all treatments (coated fillet samples), the values were significantly reduced (*p* < 0.05) compared to the control samples. In this context, Roy et al. [39] found that the composite coating based on propolis could reduce the peroxide index in coated meat products over the storage period compared to the control (uncoated samples). The XAN-EEP 2% treatment resulted in a maximal decrease in the peroxide formation, followed by XAN-EEP 1% and XAN-EEP 0%; this condition may be due to the potent antioxidant activity of XAN-EEP 2%. These results may be in line with what was mentioned by Shavisi et al. [40]. They observed that a polylactic acid (PLA) film containing ethanolic extract of propolis (EEP) reduced the peroxide value of minced beef more than the control samples that were stored in the refrigerator. fillets increased, with the control (uncoated) samples having the highest (p < 0.05) peroxide value at each interval storage period (Figure 2). The peroxide value of the control (uncoated fillet samples) increased from 2.22 to 17.32 meq peroxides/kg lipid, while during this time, the peroxide value of XAN-EEP 0%, XAN-EEP 1%, and XAN-EEP 2% increased from 2.23 to 14.55, 2.18 to 9.89, and 2.25 to 8.44 meq peroxides/kg lipid, respectively, after 20 days of chilled storage. In all treatments (coated fillet samples), the values were significantly reduced (p < 0.05) compared to the control samples. In this context, Roy et al. [39] found that the composite coating based on propolis could reduce the peroxide index in coated meat products over the storage period compared to the control (uncoated samples). The XAN-EEP 2% treatment resulted in a maximal decrease in the peroxide formation, followed by XAN-EEP 1% and XAN-EEP 0%; this condition may be due to the potent antioxidant activity of XAN-EEP 2%. These results may be in line with what was mentioned by Shavisi et al. [40]. They observed that a polylactic acid (PLA) film containing ethanolic extract of propolis (EEP) reduced the peroxide value of minced beef more than the control samples that were stored in the refrigerator.

The deterioration of the quality of fish and its products during storage is mainly due to the oxidation of lipid [11]. Lipid peroxidation is the reaction of oxygen with unsaturated lipids; hence, one of the methods that delays or prevents oxidation processes is the use of edible coatings for fish fillets [37] because the edible coatings can guarantee performance

The peroxide value (POV) is a substantial indicator of fat rancidity, but how does fat rancidity happen? Rancidity happens through the process of lipid oxidation, which is accompanied by the production of free radicals, which in turn leads to the formation of aldehydes and ketones, all of which, of course, negatively affect the quality of fish [38]. As the storage period progressed, the peroxide values in coated and uncoated mackerel tuna

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2.2.2. Oxidative Stability of Mackerel Tuna Fillets

as a low oxygen barrier [24].

Figure 2. The influence of coating treatments on peroxide values (meq/kg) in mackerel tuna fillet samples during storage at 2 °C for 20 days. Control: Uncoated mackerel tuna fillet samples (soaked samples in sterile distilled water). XAN-EEP 0%: Coated samples with xanthan containing (0%) ethanolic extract of propolis. XAN-EEP 1%: Coated samples with xanthan containing (1%) ethanolic extract of propolis. XAN-EEP 2%: Coated samples with xanthan containing (2%) ethanolic extract of propolis. a–d: Within a column, different superscripts indicate significant differences (p < 0.05). **Figure 2.** The influence of coating treatments on peroxide values (meq/kg) in mackerel tuna fillet samples during storage at 2 ◦C for 20 days. Control: Uncoated mackerel tuna fillet samples (soaked samples in sterile distilled water). XAN-EEP 0%: Coated samples with xanthan containing (0%) ethanolic extract of propolis. XAN-EEP 1%: Coated samples with xanthan containing (1%) ethanolic extract of propolis. XAN-EEP 2%: Coated samples with xanthan containing (2%) ethanolic extract of propolis. a–d: Within a column, different superscripts indicate significant differences (*p* < 0.05).

The results for TBARS which is an indicator of lipid oxidation [41] of mackerel tuna fillets coated in (Figure 3) showed a significant effect of the coating on the oxidation of mackerel fillets. During refrigeration, the TBARS values of XAN-EEP 0%, XAN-EEP 1%, and XAN-EEP 2% were significantly (*p* < 0.05) lower than the control. After 20 days of cold storage, the TBARS values of the XAN-EEP 0%, XAN-EEP 1%, and XAN-EEP 2% treatments were 2.25, 1.98, and 1.31 mg MDA/kg, respectively. The malondialdehyde (MDA) levels in the treated fillets (XAN, XAN-EEP 1%, and XAN-EEP 2%) were significantly lower (*p* < 0.05) than in the control sample (2.9 mg MDA/kg). Connell [42] also mentioned that the acceptable limit for the value of TBARS in a fish sample is in the range of 1 to 2 mg

Gels 2022, 8, 405 6 of 23

MDA/kg, and if the value exceeds this limit, an unpleasant smell of fish begins to develop. All tested samples for all treatments in our study exceeded the TBARS value limit after 20 days of storage. mg MDA/kg, and if the value exceeds this limit, an unpleasant smell of fish begins to develop. All tested samples for all treatments in our study exceeded the TBARS value limit after 20 days of storage.

The results for TBARS which is an indicator of lipid oxidation [41] of mackerel tuna fillets coated in (Figure 3) showed a significant effect of the coating on the oxidation of mackerel fillets. During refrigeration, the TBARS values of XAN-EEP 0%, XAN-EEP 1%, and XAN-EEP 2% were significantly (p < 0.05) lower than the control. After 20 days of cold storage, the TBARS values of the XAN-EEP 0%, XAN-EEP 1%, and XAN-EEP 2% treatments were 2.25, 1.98, and 1.31 mg MDA/kg, respectively. The malondialdehyde (MDA) levels in the treated fillets (XAN, XAN-EEP 1%, and XAN-EEP 2%) were significantly lower (p < 0.05) than in the control sample (2.9 mg MDA/kg). Connell [42] also mentioned that the acceptable limit for the value of TBARS in a fish sample is in the range of 1 to 2

Figure 3. The influence of coating treatments on TBARS values (MDA mg/kg) in mackerel tuna fillet samples during storage at 2 °C for 20 days. Control: Uncoated mackerel tuna fillet samples (soaked samples in sterile distilled water). XAN-EEP 0%: Coated samples with xanthan containing (0%) ethanolic extract of propolis. XAN-EEP 1%: Coated samples with xanthan containing (1%) ethanolic extract of propolis. XAN-EEP 2%: Coated samples with xanthan containing (2%) ethanolic extract of propolis. a–d: Within a column, different superscripts indicate significant differences (p < 0.05). **Figure 3.** The influence of coating treatments on TBARS values (MDA mg/kg) in mackerel tuna fillet samples during storage at 2 ◦C for 20 days. Control: Uncoated mackerel tuna fillet samples (soaked samples in sterile distilled water). XAN-EEP 0%: Coated samples with xanthan containing (0%) ethanolic extract of propolis. XAN-EEP 1%: Coated samples with xanthan containing (1%) ethanolic extract of propolis. XAN-EEP 2%: Coated samples with xanthan containing (2%) ethanolic extract of propolis. a–d: Within a column, different superscripts indicate significant differences (*p* < 0.05).

fillet samples than in others, most likely due to the antioxidants present (EEP) [40]. Additionally, the highest effects were noted with XAN-EEP at a concentration of 2%. TBARS values were significantly lower in the EEP-containing coated mackerel tuna fillet samples than in others, most likely due to the antioxidants present (EEP) [40]. Additionally, the highest effects were noted with XAN-EEP at a concentration of 2%.

TBARS values were significantly lower in the EEP-containing coated mackerel tuna

### 2.2.3. Total Volatile Basic Nitrogen (TVB-N) of Mackerel Tuna Fillet Samples 2.2.3. Total Volatile Basic Nitrogen (TVB-N) of Mackerel Tuna Fillet Samples

Amongst the important indicators of spoilage is TVB-N, which results from the degradation of proteins and non-protein nitrogen compounds as a response to bacterial activity as well as the presence of endogenous enzymes [43]. Figure 4 shows the changes in TVB-N values for all chip processors during cryogenic storage. At the start of storage (zero-time), TVB-N content ranged from 8.12 to 8.20 mg N/100 g for all mackerel tuna fillet samples. Over time TVB-N values increased for all samples, which was, of course, consistent with increases in pH values during later stages of storage. The results of our study are in line with those obtained by Yu et al. [44]. On the 20th day, TVB-N values for the control, XAN-EEP 0%, XAN-EEP 1%, and XAN-EEP 2% were 50.19, 42.15, 27.14, and 22.87 Amongst the important indicators of spoilage is TVB-N, which results from the degradation of proteins and non-protein nitrogen compounds as a response to bacterial activity as well as the presence of endogenous enzymes [43]. Figure 4 shows the changes in TVB-N values for all chip processors during cryogenic storage. At the start of storage (zero-time), TVB-N content ranged from 8.12 to 8.20 mg N/100 g for all mackerel tuna fillet samples. Over time TVB-N values increased for all samples, which was, of course, consistent with increases in pH values during later stages of storage. The results of our study are in line with those obtained by Yu et al. [44]. On the 20th day, TVB-N values for the control, XAN-EEP 0%, XAN-EEP 1%, and XAN-EEP 2% were 50.19, 42.15, 27.14, and 22.87 mg N/100 g, respectively. Thirty-five to forty milligrams of nitrogen per one hundred grams is the acceptable limit for TVB-N values in fresh fish, as reported by Connell [42]. Grigorakis et al. [45] suggested that the acceptable limit for TVB-N values in chilled sea bass is 19–20 mg N/100 g. Twenty-five to thirty-five of nitrogen per one hundred grams was considered a limit for mackerel tuna fillet damage in our study. The edible films may extend the shelf life of the fish fillet by reducing gas permeability and penetration, especially oxygen permeability, thereby limiting bacterial growth and activity [46].

Figure 4. The influence of coating treatments on TVB-N values (mg/100 g) in mackerel tuna fillet samples during storage at 2 °C for 20 days. Control: Uncoated mackerel tuna fillet samples (soaked samples in sterile distilled water). XAN-EEP 0%: Coated samples with xanthan containing (0%) ethanolic extract of propolis. XAN-EEP 1%: Coated samples with xanthan containing (1%) ethanolic extract of propolis. XAN-EEP 2%: Coated samples with xanthan containing (2%) ethanolic extract of propolis. a–d: Within a column, different superscripts indicate significant differences (p < 0.05). **Figure 4.** The influence of coating treatments on TVB-N values (mg/100 g) in mackerel tuna fillet samples during storage at 2 ◦C for 20 days. Control: Uncoated mackerel tuna fillet samples (soaked samples in sterile distilled water). XAN-EEP 0%: Coated samples with xanthan containing (0%) ethanolic extract of propolis. XAN-EEP 1%: Coated samples with xanthan containing (1%) ethanolic extract of propolis. XAN-EEP 2%: Coated samples with xanthan containing (2%) ethanolic extract of propolis. a–d: Within a column, different superscripts indicate significant differences (*p* < 0.05).

mg N/100 g, respectively. Thirty-five to forty milligrams of nitrogen per one hundred grams is the acceptable limit for TVB-N values in fresh fish, as reported by Connell [42]. Grigorakis et al. [45] suggested that the acceptable limit for TVB-N values in chilled sea bass is 19–20 mg N/100 g. Twenty-five to thirty-five of nitrogen per one hundred grams was considered a limit for mackerel tuna fillet damage in our study. The edible films may extend the shelf life of the fish fillet by reducing gas permeability and penetration, espe-

cially oxygen permeability, thereby limiting bacterial growth and activity [46].

In comparison to the other treatments, the propolis-treated mackerel tuna fillets had the lowest TVB-N values. Similar observations were obtained by Bazargani-Gilani et al. [47]. These results can be interpreted based on the ability of propolis to inhibit microbial activity, including the inhibition of bacteria responsible for the deamination reaction of non-protein nitrogen (NPN) components [46]. In comparison to the other treatments, the propolis-treated mackerel tuna fillets had the lowest TVB-N values. Similar observations were obtained by Bazargani-Gilani et al. [47]. These results can be interpreted based on the ability of propolis to inhibit microbial activity, including the inhibition of bacteria responsible for the deamination reaction of non-protein nitrogen (NPN) components [46].

#### 2.2.4. K-Value of Mackerel Tuna Fillet Samples 2.2.4. *K*-Value of Mackerel Tuna Fillet Samples

Endogenous biochemical changes occur in fish muscle during postmortem fish storage, among which is nucleotide degradation [48]. Calculation of the contents of ATP and its associated degradation products is an effective indicator for monitoring the freshness of fish fillets [47]. Changes in the K value during cryogenic storage of mackerel tuna fillets are shown in Figure 5. The initial K-values of the control and treated mackerel tuna samples ranged from 15.31 to 16.82%. K-values of uncoated (control) and coated mackerel tuna fillet samples increased significantly (p < 0.05) with storage time. Additionally, the treatments illustrated significantly lower K-values (p < 0.05) than the control sample. According to previous studies, the rejection level of the K-value was close to 60% [49]. Control exceeded this limit on the 10th day (68.14%), while XAN-EEP 0% exceeded this limit Endogenous biochemical changes occur in fish muscle during postmortem fish storage, among which is nucleotide degradation [48]. Calculation of the contents of ATP and its associated degradation products is an effective indicator for monitoring the freshness of fish fillets [47]. Changes in the K value during cryogenic storage of mackerel tuna fillets are shown in Figure 5. The initial K-values of the control and treated mackerel tuna samples ranged from 15.31 to 16.82%. K-values of uncoated (control) and coated mackerel tuna fillet samples increased significantly (*p* < 0.05) with storage time. Additionally, the treatments illustrated significantly lower K-values (*p* < 0.05) than the control sample. According to previous studies, the rejection level of the K-value was close to 60% [49]. Control exceeded this limit on the 10th day (68.14%), while XAN-EEP 0% exceeded this limit on the 15th day (72.19%), and both XAN-EEP 1% and XAN-EEP 2% exceeded this limit on the 20th day (68.04, 59.99%).

Figure 5. The influence of coating treatments on K values (%) in mackerel tuna fillet samples during storage at 2 °C for 20 days.Control: Uncoated mackerel tuna fillet samples (soaked samples in sterile distilled water). XAN-EEP 0%: Coated samples with xanthan containing (0%) ethanolic extract of propolis. XAN-EEP 1%: Coated samples with xanthan containing (1%) ethanolic extract of propolis. XAN-EEP 2%: Coated samples with xanthan containing (2%) ethanolic extract of propolis. a–d: Within a column, different superscripts indicate significant differences (p < 0.05). **Figure 5.** The influence of coating treatments on K values (%) in mackerel tuna fillet samples during storage at 2 ◦C for 20 days. Control: Uncoated mackerel tuna fillet samples (soaked samples in sterile distilled water). XAN-EEP 0%: Coated samples with xanthan containing (0%) ethanolic extract of propolis. XAN-EEP 1%: Coated samples with xanthan containing (1%) ethanolic extract of propolis. XAN-EEP 2%: Coated samples with xanthan containing (2%) ethanolic extract of propolis. a–d: Within a column, different superscripts indicate significant differences (*p* < 0.05).

on the 15th day (72.19%), and both XAN-EEP 1% and XAN-EEP 2% exceeded this limit on

#### 2.3. Microbiological Analyses of Mackerel Tuna Fillets *2.3. Microbiological Analyses of Mackerel Tuna Fillets*

Generally, propolis' antimicrobial characteristics may be responsible for the inhibition of microbial growth in stored fish fillets [50]. Specifically, the following sections will focus on the changes in each microbial group in mackerel tuna fillet samples. Generally, propolis' antimicrobial characteristics may be responsible for the inhibition of microbial growth in stored fish fillets [50]. Specifically, the following sections will focus on the changes in each microbial group in mackerel tuna fillet samples.

#### 2.3.1. Total Viable Count (TVC) 2.3.1. Total Viable Count (TVC)

whole storage period.

the 20th day (68.04, 59.99%).

Changes in total viable count (TVC) of mackerel tuna fillet samples during refrigerated storage are shown in Figure 6A. The initial TVC (log10 CFU/g) of all samples, including the control and treatments, ranged from 2.5 to 3.0. Compared to the values reported by Yu et al. [44] for grass carp fillets (4.90 log10 CFU/g), the values obtained in our study were lower. The reason for this may be attributed to individual differences or the handling of the fish during processing. The lower initial TVC for coated mackerel tuna fillets indicated that XAN-EPP coating reduced the microbial population. According to the International Commission on Microbiological Specifications for Foods (ICMSF) [51], the maximum allowable TVC is 7.0 log10 CFU/g. Based on that and looking at the results of our study, it was found that over the storage period, there was a noticeable increase (p < 0.05) in TVC for untreated samples compared to treated samples, until the untreated samples (control group) exceeded the permissible limit of TVC after 11 days. XAN-EEP 0% samples have exceeded the TVC limit after 16 days. For mackerel tuna fillets treated with 1% and 2% ethanolic extract of propolis (EEP), TVC was below the limit level during the Changes in total viable count (TVC) of mackerel tuna fillet samples during refrigerated storage are shown in Figure 6A. The initial TVC (log<sup>10</sup> CFU/g) of all samples, including the control and treatments, ranged from 2.5 to 3.0. Compared to the values reported by Yu et al. [44] for grass carp fillets (4.90 log<sup>10</sup> CFU/g), the values obtained in our study were lower. The reason for this may be attributed to individual differences or the handling of the fish during processing. The lower initial TVC for coated mackerel tuna fillets indicated that XAN-EPP coating reduced the microbial population. According to the International Commission on Microbiological Specifications for Foods (ICMSF) [51], the maximum allowable TVC is 7.0 log<sup>10</sup> CFU/g. Based on that and looking at the results of our study, it was found that over the storage period, there was a noticeable increase (*p* < 0.05) in TVC for untreated samples compared to treated samples, until the untreated samples (control group) exceeded the permissible limit of TVC after 11 days. XAN-EEP 0% samples have exceeded the TVC limit after 16 days. For mackerel tuna fillets treated with 1% and 2% ethanolic extract of propolis (EEP), TVC was below the limit level during the whole storage period.

(A)

**Figure 6.** *Cont*.
